Figure 1.

Principle of interference-free quantification based on the TMT^C cluster. (A) A peptide (brown) is labeled with TMT-131 or TMT-126 reagents, and both forms are mixed in a ratio of 1:1. Asterisks on the TMT structures indicate heavy isotopes (¹³C or ¹⁵N). An interfering peptide (blue) is coeluting with the peptide of interest within the isolation window for the MS2 spectrum. When peptide ions are subjected to higher-energy collisional dissociation (HCD), the TMT tags fragments at two positions, as indicated with dotted lines. (B) Typically, the MS2 spectrum contains both the reporter ions (left) and the TMT^C cluster (right), which contains most of the mass-balancing part of the TMT tag. Quantification based on the reporter ions is inaccurate as interfering peptides produce reporter ions of identical mass. In contrast with high-resolution MS2 spectra, the TMT^C ions for the peptide of interest (and any coeluting peptides) can typically be distinguished, and therefore the TMT^C ion cluster comprises accurate quantitative information. Note that this approach can be used to quantify multiple peptides in a single MS2 spectrum; e.g., in the toy example shown here the brown peptide ratio for the TMT-126 and TMT-131 channels would be 1:1, whereas the blue peptide ratio would be 0:1.